1. Field of the Invention
This invention relates to antimicrobial compositions which are capable of activating or supplementing naturally occurring peroxidase systems. In particular, compositions which are stable during preparation and after packaging in environments which contain some oxygen, methods of making such compositions, and methods of use are disclosed.
2. Art Background
There is general acceptance as to the etiology of dental caries and periodontal disease in that microflora found in the oral environment are capable of accumulating upon oral surfaces and in unexposed pockets, thriving and producing damaging metabolites in the absence of proper dental hygiene. Colonies of microbes, undisturbed for even short periods of time, are able to aggressively adhere to the surface of enamel, establishing a foothold for further colony growth. Many of the bacterial types commonly found in the mouth secrete polysaccharides such as glucans and dextrans, which form a supportive matrix and thus provide a more mechanically stable environment for further proliferation. Subgingivally, undisturbed colonies of aerobic and anaerobic bacteria can establish similar polysaccharide matrices, in addition to pocket-type formations.
These polysaccharide matrices, together with the thriving microflora contained therein, make up what is commonly referred to as plaque. The first stages in plaque formation occur almost immediately after an enamel surface is scraped, cleaned and polished in dental office tooth cleaning procedures. As colony numbers increase, and the structural integrity of the surrounding polysaccharide matrix evolves, plaque becomes a potential source of bacterial metabolites such as lactic acid. In intimate contact with the enamel surface, acidic plaque metabolites are thus capable of lowering the pH of the enamel surface to a point at which demineralization of the hydroxyapatite can occur. Such demineralization is known to be the cause of tooth decay, also known as caries. Subgingivally, plaque and pocket colonies are known to cause demineralization of both enamel and periodontal bone structure. Gingivitis and periodontitis, infection and irritation of the soft tissues surrounding the teeth, are other clinical manifestations of subgingival plaque and pocket colony proliferation.
One approach taken to decrease caries is by limiting the demineralization of enamel and bone through drinking water fluoridation. It has been shown that the fluoride provided by drinking water (and to a more limited extent, through diet) is capable of being incorporated into hydroxyapatite, the major inorganic component of enamel and bone. Fluoridated hydroxyapatite is less susceptible to demineralization by acids and is thus seen to resist the degradative forces of acidic plaque and pocket metabolites. In addition, fluoride ion concentration in saliva is increased through consumption of fluoridated drinking water. Saliva thus serves as an additional fluoride ion reservoir; in combination with buffering salts naturally found in salivary fluid, fluoride ions are actively exchanged on the enamel surface, further offsetting the effects of demineralizing acid metabolites.
A large body of data indicates that drinking water fluoridation leads to a statistically significant decrease in DMF (decayed, missing, and filled) teeth for a broad range of populations studied. Smaller, less significant effects are seen in fluoridated drinking water studies which examine changes in periodontal health. Positive periodontal effects are thought to arise through the antimicrobial effects of increased fluoride ion concentration in saliva.
However, notwithstanding the established benefits of fluoride treatment of teeth, fluoride ion treatment can result in the mottling of teeth, whether administered systemically through drinking water or topically applied. This effect is known to be both concentration related and patient-specific. In addition, the toxicology of fluoride has recently come under closer scrutiny, although there is no clear answer as to its long term effect on human health. However, for the time being, drinking water fluoridation is believed to serve a wider public good, and its effect on the dental health of populations the world over are pronounced.
Another approach to limiting the proliferation of microflora in the oral environment is through the topical or systemic application of broad-spectrum antibacterial compounds. By killing large numbers of oral microflora, it is postulated, plaque and pocket accumulation, together with their damaging acidic metabolite production, can be reduced or eliminated. The major drawback to such an approach is that there are a wide variety of benign or beneficial strains of bacteria found in the oral environment, which are killed by the same antibacterial compounds in the same manner as the harmful strains. In addition, such treatment with antibacterial compounds may select for certain bacteria and most fungi, which may then be resistant to the antibacterial compound administered, and thus proliferate, unrestrained by the symbiotic forces of a properly balanced microflora population. Such a selected proliferative process leads to yet another clinical problem which must then be addressed with other antimicrobial strategies. Thus, the application or administration of broad-spectrum antibiotics is ill-advised, except in preventative or palliative clinical situations such as oral surgery, severe periodontitis, and immune dysfunction diseases.
Less potent and more selective antimicrobial compounds have been devised, which, when applied topically, have achieved varying degrees of success in checking the growth of harmful oral microorganisms. Of particular interest and relevance to the subject matter of the present invention are those approaches which attempt to activate or supplement the antimicrobial potential of saliva.
Saliva is known to contain a variety of immunoglobulin and non-immunoglobulin antibacterial compounds as a defense against the proliferation of harmful pathogens. Such non-immunoglobulin proteins include lysozyme, lactoferrin and salivary peroxidase. These proteins, or ones similar in function, are found in virtually all mammalian mucosal secretions, providing a first line of defense against pathogenic organisms which would otherwise rapidly proliferate in such warm, moist environments. The enzyme salivary peroxidase, or SPO, functions by utilizing hydrogen peroxide (produced and excreted primarily by certain bacteria as a metabolite, but found also in newly expressed saliva) to oxidize a pseudohalide ion found in saliva, thiocyanate (SCN--), to produce a potent bacteriostatic agent, hypothiocyanite ion (OSCN--). Hypothiocyanite ion and its corresponding acid, hypothiocyanous acid (herein referred to collectively as hypothiocyanite) are able to inhibit the growth of a wide variety of harmful pathogens found in the oral environment. Depending upon the concentration of hypothiocyanite in the saliva, the salivary peroxidase system can either merely inhibit microbial metabolism or actually kill the organism. In general, it has been shown that concentrations of hypothiocyanite greater than about 100 micromoles/liter are sufficient to inhibit the metabolism of plaque bacteria.
Since the salivary peroxidase system, and thus the production of hypothiocyanite, is dependent upon the availability of hydrogen peroxide, various prior art attempts to provide sufficient hydrogen peroxide to activate or supplement the SPO system have been made. Conversely, since SPO begins to show inhibition by concentrations of hydrogen peroxide greater than about 1 millimole/liter, an effective SPO activation mechanism should not provide or accumulate peroxide molarities much higher than this. Direct inclusion of hydrogen peroxide in a mouth rinse composition at these low concentrations has been shown to activate the SPO system for short periods of time (Mansson-Rahemtulla, et al., J. of Dental Res. 62(10): 1062-1066). Another prior art attempt to generate hydrogen peroxide in situ comprised including an oxidoreductase enzyme, such as glucose oxidase, in a dentifrice (Hoogendorn, et al., U.S. Pat. Nos. 4,150,113 and 4,178,519). The glucose oxidase thus provided would, upon oral application, react with glucose present in saliva and in plaque interstitial fluid to produce hydrogen peroxide at low concentrations. Since this approach was dependent upon the availability of glucose in the mouth, a more reproducible and predictable route to enzymatic hydrogen peroxide production was then taken by the present inventor and others by including both glucose oxidase and beta-D-glucose within a dentifrice composition. (U.S. Pat. No. 4,537,764). Beta-D-glucose is the anomer of glucose for which glucose oxidase is specific; in aqueous solution, glucose will mutorotate rapidly to form a mixture of approximately 65% beta-D-glucose and 35% alpha-D-glucose. In order to prevent instability and premature enzyme/substrate interaction the amount of water in the composition had to be limited to less than 10 percent. Upon use of this dentifrice composition, additional water present (from saliva and from water added in the course of normal toothbrushing procedures) would dilute the composition to a water content of greater than 10 percent, thus allowing reaction between glucose oxidase and glucose to ensue. The hydrogen peroxide thus created as a product of reaction would activate the salivary peroxidase system in saliva, producing hypothiocyanite.
Later attempts were made to provide a dentifrice composition containing a complete system of components capable of generating hypothiocyanite in situ (U.S. Pat. Nos. 4,564,519 and 4,578,265). An oxidoreductase enzyme together with its corresponding substrate were combined in a single dentifrice composition with a peroxidase enzyme and a thiocyanate salt, thus providing a method of producing hypothiocyanite independent of fluctuations in salivary glucose, salivary peroxidase and salivary thiocyanate ion. Again, stability of such dentifrice compositions containing a complete enzymatic system capable of producing hypothiocyanite could only be maintained by formulating with less than about 10 percent water. Similarly, the reaction sequence was started by dilution of the dentifrice during toothbrushing.
There are numerous other examples in the prior art of attempts to provide a stable enzymatic dentifrice containing both an oxidoreductase enzyme and its specific substrate for the purpose of producing hydrogen peroxide. Stability of such prior art compositions has been achieved by either limitations placed on the amount of water contained within the composition or by physically separating (through microencapsulation, U.S. Pat. No. 4,978,528) the oxidoreductase enzyme from its specific substrate.
In light of the foregoing description, it would be advantageous to provide a stable, aqueous enzymatic composition capable of supplementing, or, in the presence of saliva or other humoral secretions, activating the peroxidase system in such a fashion that hypothiocyanite ions (OSCN--) are produced in excess of about 100 micromoles/liter/minute in vitro or in vivo.
In addition to the salivary peroxidase system, other natural antimicrobial systems can be found in the humoral secretions of all mammals. These antimicrobial systems consist of a peroxidase enzyme (for example, salivary peroxidase found in saliva and cervical peroxidase found in vertical fluid) together with an oxygen acceptor such as thiocyanate or iodide ion. Both components are normally found in the secretions themselves, and the addition of exogenous hydrogen peroxide activates the system and produces potent antimicrobial species, such as hypothiocyanite and hypoiodite (OSCN-- and OI--, respectively).
As described in my prior application referenced above, aqueous compositions capable of producing hydrogen peroxide enzymatically through the action of an oxidoreductase enzyme must be manufactured so as to contain a low level of dissolved oxygen in order to prevent the premature formation of hydrogen peroxide prior to the time of intended use. Such compositions must also be packaged in an essentially anaerobic fashion so as to exclude oxygen during storage. These requirements are the subject of my prior application referred to above.
It would also be advantageous to provide a stable, aqueous enzymatic composition capable of producing or, in the presence of saliva, leading to the production of hypothiocyanite, irrespective of the composition's water content or the amount of water available for dilution upon use. Additionally, formulating latitude and economy would greatly benefit from such aqueous enzymatic dentifrice compositions produced and stabilized without regard to the amount of water contained within the formulation. It would also be advantageous to provide such a composition which may be formulated in the presence of oxygen without having to be particularly careful about removing and/or replacing the oxygen which is present until it is packaged.
It would be of additional utility to provide a method of manufacturing a stable, aqueous enzymatic composition which contains both an oxidoreductase enzyme and its specific substrate, yet prevents hydrogen peroxide accumulation prior to its intended use.